Generation and effect of photo-induced radicals on cross-linking, color change, and isomerization in formulations of peptides and proteins

Protein biotherapeutics, especially monoclonal antibodies (mAbs), have been on the rise due to their high efficacy, potency, and low toxicity. They are mainly used for the treatment of cancer, autoimmune diseases, infectious diseases, and organ transplantation. While mAbs are well-tolerated by patients and have fewer adverse effects, their stability can be challenged by physical and chemical degradation. Hence, they are formulated with buffers and other excipients such as polysorbate 80 (PS80), which help to maintain the pH of the formulation and prevent aggregation, respectively. However, when the drug product is exposed to light, heat, or contains metal impurities, they are likely to undergo degradation. Some of the reactive amino acids of the proteins such as Trp, Tyr, His, Met, and Cys residues are more prone to photooxidation. Thus, we have investigated the effect of both UV and visible light on peptides, and proteins and further looked into the role of protein- and buffer-derived radicals on degradation of PS80. We explored the role of methionine sulfur cation, generated by photosensitization of 4-carboxybenzophenone in the Met-Xn-His-containing peptides (n = 0 - 2), a common sequence present in the biotherapeutics such as mAb and human parathyroid hormone. Here, we report on the formation of novel photo-oxidation products and cross-links between Met oxidation product(s) and a neighboring histidine residue. Mechanisms for the formation of these products will be proposed. Specifically, the formation of cross-links is hypothesized to involve photo-oxidation of Met to an aspartate semialdehyde, followed by reaction with the imidazole side chain of His, and elimination of water. When a full mAbZ (mAb obtained from AstraZeneca) was exposed to visible light, discoloration of the solution was observed. The chromophoric product responsible for the color change was identified using a model Trp-containing compound, N-acetyl-L-tryptophan amide (NATA). The product was identified as NATA-33, a conjugated product formed after loss of 33 Da from NATA, by mass spectrometry and NMR. The mAbZ formulation contains polysorbate 80 (PS80) that has unsaturated fatty acids such as oleic acid and linoleic acid. Hence, we address the question of how protein-derived radicals may affect the composition of PS80. Isobaric products of PS80 were identified by means of mass spectrometry, suggesting cis/trans isomerization of unsaturated fatty acids of PS80. This mechanism was confirmed by the analysis of isolated fatty acids, demonstrating, e.g., the conversion of oleic acid to elaidic acid. Another commonly used excipient in mAb biotherapeutics is citrate buffer. In the presence of metal impurities such as iron, citrate buffer and iron can generate citrate-derived degradants such as carbon dioxide radical anion after exposure to UV-A light. Such radical anion can donate an electron to a disulfide bond and form a thiyl radical, which can also induce cis/trans isomerization of unsaturated fatty acids of PS80. Therefore, photo-induced radicals and radical ions may lead to cross-linking of amino acids, color change in concentrated mAbs, and cis/trans isomerization of PS80

A candidate multimodal functional genetic network for thermal adaptation

Vertebrate ectotherms such as reptiles provide ideal organisms for the study of adaptation to environmental thermal change. Comparative genomic and exomic studies can recover markers that diverge between warm and cold adapted lineages, but the genes that are functionally related to thermal adaptation may be difficult to identify. We here used a bioinformatics genome-mining approach to predict and identify functions for suitable candidate markers for thermal adaptation in the chicken. We first established a framework of candidate functions for such markers, and then compiled the literature on genes known to adapt to the thermal environment in different lineages of vertebrates. We then identified them in the genomes of human, chicken, and the lizard Anolis carolinensis, and established a functional genetic interaction network in the chicken. Surprisingly, markers initially identified from diverse lineages of vertebrates such as human and fish were all in close functional relationship with each other and more associated than expected by chance. This indicates that the general genetic functional network for thermoregulation and/or thermal adaptation to the environment might be regulated via similar evolutionarily conserved pathways in different vertebrate lineages. We were able to identify seven functions that were statistically overrepresented in this network, corresponding to four of our originally predicted functions plus three unpredicted functions. We describe this network as multimodal: central regulator genes with the function of relaying thermal signal (1), affect genes with different cellular functions, namely (2) lipoprotein metabolism, (3) membrane channels, (4) stress response, (5) response to oxidative stress, (6) muscle contraction and relaxation, and (7) vasodilation, vasoconstriction and regulation of blood pressure. This network constitutes a novel resource for the study of thermal adaptation in the closely related nonavian reptiles and other vertebrate ectotherms

End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions